Method and system for treating wastewater and sludges by optimizing sCO2 for anaerobic autotrophic microbes

ABSTRACT

The present invention describes a method of optimizing CO 2  concentration to increase the specific growth rate of Anammox bacteria and methanogens in wastewater and sludge treatment, as well as novel systems and methods of treating wastewater and sludge. The specific growth rate or doubling time of the Anammox bacteria and methanogens were determined to be sensitive to dissolved CO 2  concentration. Optimizing dissolved CO 2  concentration increases the specific growth rate of the Anammox bacteria, which may be used as an alternative biological nitrogen removal process for the treatment of domestic wastewater. In the method and system of treating sludge, the CO 2  stripper returns biogas with low CO 2  concentration to the headspace of an anaerobic digester in order to lower the headspace CO 2  concentration and therefore, the soluble CO 2  concentration. The lower soluble CO 2  concentration increases the specific growth rate of the methanogens for a more efficient anaerobic digestion process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior filed InternationalApplication, Serial Number PCT/US2012/025237 filed Feb. 15, 2012, whichclaims priority to U.S. Provisional Application No. 61/443,089 entitled“Method of Optimizing CO₂ Levels to Increase Bacteria Levels in SludgeTreatment”, filed Feb. 15, 2011 and U.S. Provisional Application No.61/487,504 entitled “Optimization of Dissolved Carbon Dioxide forAnaerobic Autotrophic Bacteria and Archaea”, filed May 18, 2011, thecontents of each of which are hereby incorporated by reference into thisdisclosure.

FIELD OF INVENTION

This invention relates to water treatment. Specifically, the inventiondiscloses a method of optimizing CO₂ levels to increase the specificgrowth rate of Anammox bacteria for efficient nitrogen removal fromwastewater and methanogens for rapid anaerobic digestion of sewagesludges and other biodegradable solids. In addition, novelconfigurations of wastewater treatment systems and anaerobic digestersystems are provided that utilize CO₂ stripping equipment to provideoptimal growth conditions for Anammox and methanogens with respect tosoluble CO₂ concentrations.

BACKGROUND OF THE INVENTION

For the past century, environmental engineers have been using theactivated sludge system and anaerobic digestion to successfully treatmunicipal wastewater (Metcalf & Eddy (2003). Wastewater Engineering:Treatment and Reuse. New York, N.Y., McGraw-Hill). In the United States,the nitrification process in the activated sludge system of public ownedtreatment works (POTWs) is very expensive with capital costs of theaeration basin alone valued at $26B and annual electricity costs of$335M (EPA (1996). Clean Watersheds Needs Survey (CWNS). W. D.C. Officeof Water; Goldstein, R. and W. Smith (2002). Water & Sustainability(Volume 4): U.S. Electricity Consumption for Water Supply &Treatment—The Next Half Century. Palo Alto, EPRI; EPA (2007). BiologicalRemoval Processes and Costs. W. D.C. Office of Water). More recently,the United States Environmental Protection Agency has proposed astricter effluent nutrient limit (Total N of 0.82-1.73 mg/L and Total Pof 0.069-0.415 mg/L) for Florida POTWs that is estimated to cost$24.4-50.7B in capital investment and increase annual operating expensesby $0.4-1.3B (Oskowis, J. (2009). Re: Numeric Nutrient Criteria CostImplications for Florida POTW's G. C. Crist). Over half of the capitalinvestment will upgrade the existing activated sludge system to anadvanced biological nutrient removal (BNR) system. It is anticipatedthat the numerous extended aeration plants used for secondary treatmentof wastewater in FL will be upgraded to Bardenpho 5-stage (BP5) systemsor other advanced BNR system for biological nitrogen and phosphorusremoval. The BP5 upgrade of the extended aeration plants will mostlikely not include the introduction of a primary clarifier and instead,the process will be operated with an elevated integrative resourcerecovery (IRR). The impact of the IRR on the microbial ecology of BNRsystems has not been reported.

Researchers used empirical studies to identify key operating parametersfor these systems to ensure effective performance. In order to increasethe protection of surface waters from excessive oxygen demand fromtreated wastewater, engineers have modified the simple aeration basin toinclude anoxic and anaerobic zones and recycled nitrate-rich wastewaterand anaerobic digester supernatant to promote biological nutrientremoval. The anoxic zones allow for nitrogen removal by providingconditions with no dissolved oxygen and high nitrate levels fordenitrification of nitrate rich wastewater. The anaerobic zones arenecessary for phosphorous removal because anaerobic (no dissolved oxygenor nitrate) conditions are necessary for the phosphorous accumulatingorganisms (PAO) to release phosphorous and take up volatile fatty acids.These anaerobic zones are not used to generate methane.

The activated sludge system designs and operations are dependent onproviding adequate biomass concentration in the aeration tank,environmental conditions for the biomass, and adequate time for thebioreaction. Carbonaceous biochemical oxygen demand (BOD) and ammoniumare consumed as substrate by the heterotrophic and nitrifying bacteria,respectively. The conventional nitrification process requires a lengthysolids retention time (SRT), which prevents the washout of theslow-growing aerobic autotrophic microbes.

In addition to the aerobic, autotrophic bacteria (nitrifying bacteria),the growth of anaerobic, autotrophic bacteria (Anammox bacteria andothers) and Archaea (methanogens) are also sensitive to the dissolvedCO₂ concentration. Like the aerobic, autotrophic bacteria, the Andrew'sequation describes this sensitivity of their specific growth rate to thedissolved CO₂ concentration. The anaerobic, autotrophic Archaea arethought to have a conserved metabolism with respect to the evolutionarytime-scale, which may explain the similar Andrew's equation predictionsfor the optimal dissolved CO₂ concentration for both bacteria andArchaea. In other words, the similarities in the predictions of theAndrew's equation for the optimal dissolved CO₂ concentration for thespecific growth rate of microbes may be the result of a common ancestorto both bacteria and archaea. This would predict that all autotrophicbacteria and archaea have similar autotrophic metabolism and dissolvedCO₂ sensitivities. This new knowledge can be useful for the improvementof the current practice for the treatment of wastewater (i.e., Anammoxfor nitrogen removal), sludges (i.e., methanogenesis for anaerobicdigestion), and contaminated soils (i.e., dechlorinating bacteria andmethanogens for complete mineralization of chlorinated organics) whereanaerobic, autotrophic microbes are recognized as the rate-limitingstep. In addition, landfill operation (i.e., methanogens) and biogenicmethane production of coal beds (i.e., methanogens) may also benefit byoptimization of dissolved CO₂ concentration.

Anammox bacteria can be used in sludge treatment systems to treatanaerobic digester sludge supernatant that consists of high levels ofammonium. Typically, the SHARON reactor is used to oxidize one half ofthe ammonium to nitrite and the blend of ammonium and nitrite is fed tothe Anammox reactor. Researchers have reported that the Anammox bacteriahave a very long doubling time of 12 days. However, the operation of theAnammox reactor is at 5% CO₂ in the headspace and controlled temperature(35° C.). This results in an elevated dissolved CO₂ concentration, whichinhibits the growth of these anaerobic, autotrophic bacteria. Operationat much lower and optimal dissolved CO₂ concentration will reduce thedoubling time to a few hours, which improve the performance of thesebioreactors and may offer the opportunity for utilization of the Anammoxbacteria for the treatment wastewater at ambient temperatures. Thisapproach would reduce the capital and operating costs for BNR systems.

Current operation of anaerobic digesters for the bioconversion oforganic solids to biogas (i.e., methane and carbon dioxide) exposes thebiomass to the biogas. This exposure to the biogas controls thedissolved carbon dioxide based on Henry's constant, temperature, and thepartial pressure of carbon dioxide (pCO₂) in the biogas. The carbondioxide concentration of the biogas for anaerobic digesters is typicallybetween about 35 to about 50%. This range of gas phase carbon dioxideresults in an elevated dissolved carbon dioxide concentration, whichinhibits the growth of the methanogens. The methanogens are consideredto be the rate-limiting step in anaerobic digesters with specific growthrate of 0.35 d⁻¹ used for the design of the bioreactors. Anaerobicdigestion of sludge typically requires the use of large holding tankssized to accommodate enough sludge to account for a 20-day hydraulicretention time, which is required to maintain adequate biomass of slowlygrowing methanogens.

With proper control of the gas phase carbon dioxide level in the biogasexposed to the biomass, the specific growth rate of the methanogens canbe increased substantially. For example, operation at 5% CO₂ in theheadspace for the direct production of biomethane (95-98% CH₄; 2-5% CO₂)increases the specific growth to about 2.36-2.92 d⁻¹ compared to about0.30-0.38 d⁻¹ for conventional systems. The faster specific growth rateof the methanogens will allow for the design and operation of smalleranaerobic digesters for equivalent organic loading rates. With a safetyfactor of 5, conventional anaerobic digesters are operated at a minimumsolids retention time (SRT) of 15 days, which corresponds to 0.31 d⁻¹for methanogens. With this invention and same safety factor, the minimumsolids retention time for an anaerobic digester generating biomethane isabout 1.71-2.12 days. This translates into a reactor that is betweenabout 11-14% of the size of the conventional anaerobic digester used forthe treatment of municipal sewage sludges. Operation at headspace CO₂concentrations lower than 5% will further increase the specific growthrate of the methanogens, but it may not be economically feasible due tothe capital costs of the CO₂ stripper and solids pretreatmenttechnologies required for improved biodegradability of sewage solids.For operation at lower SRT, pretreatment of feed sludges or otherorganic solids that have low hydrolysis rates may be necessary. Forexample, the minimum hydrolysis rates for primary and waste activatedsludge are 0.4 and 0.15 d⁻¹, respectively. It is unclear whether therapid rate of methanogenesis from the optimization of the soluble CO₂concentration may improve the rates of hydrolysis and subsequentfermentation by reducing the concentration of soluble and gaseousintermediates. A number of physical process technologies are availablethat treat the sewage sludges at high temperatures and/or pressures inorder to generate soluble organics.

Alternatively, an existing anaerobic digester operating at an SRT of 15days could be fed much higher organic loading rates than is recommendedwithout inhibition of performance. This higher organic loading ratecould be accomplished by either thickening the sewage sludges or addingother types of organic solids, such as food or paper wastes. Theseorganic solids, especially the paper wastes, may require pretreatment toimprove their biodegradability.

A biological approach improves the degradability of the sewage sludgesprior to methanogenesis by the use of a thermophilic (55° C.) anaerobicreactor (acidogenic bioreactor) with a low SRT (2-3 days) for theexpress purpose of generating high levels of volatile fatty acids fromthe hydrolysis and fermentation of the sewage sludges. The Glendalewastewater treatment plant at Lakeland, Fla. utilizes this approach (2.1days SRT in the thermophilic, acidogenic bioreactor) and generates lowrates of biogas (37% CH₄). Despite the low SRT, the thermophilicmethanogens are still present and active in the system. The elevated CO₂concentration prevents the thermophilic methanogens from growing atfaster rates. Operation at a lower headspace CO₂ concentration willincrease the specific growth rate of the thermophilic methanogens, whichmay eliminate the need for the downstream mesophilic anaerobic digester(methanogenic bioreactor).

Optimizing the specific growth rate of the Anammox bacteria andmethanogens by controlling the soluble CO₂ concentration also has greatimplications in the biological treatment of wastewater and sludges. Forbiological nitrogen removal, the control of soluble CO₂ concentration isa new tool for designers of wastewater treatment systems that promiselower capital and operating costs. Rapid growth by Anammox bacteria bycontrol of soluble CO₂ concentration makes efficient nitrogen removalfrom wastewater at ambient conditions a possibility. The inventor hasalso discovered that the gas phase CO₂ concentration can be optimizedsuch that the rate of biogas formation is improved in the anaerobicdigestion of sewage sludges. Increasing the specific growth rate of themethanogens by control of the soluble CO₂ concentration by stripping CO₂from the collected biogas decreases the required solids retention timeand in turn smaller digesters can be used which will save on capitalcosts. Heating the contents of the anaerobic digesters may not benecessary, if higher specific growth rates of methanogens are exhibiteddue to control of the soluble CO₂ concentration, which will loweroperating costs and make more biomethane available for other uses.

SUMMARY OF INVENTION

The growth sensitivity of anaerobic autotrophic microbes to dissolvedCO₂ has not previously been studied. The present invention describes amethod of optimizing the soluble carbon dioxide (sCO₂) concentration ina wastewater treatment system, which increases the specific growth rateof Anammox bacteria and methanogens.

The rapidly growing Anammox bacteria can be used as an alternative tothe nitrifying step currently being used in wastewater treatmentfacilities. A method and system of treating wastewater in which anAnammox reactor is added to the system and the sCO₂ concentration isoptimized is presented.

Laboratory-scale anaerobic digesters demonstrated the impact of loweringthe gas phase concentration of carbon dioxide on the biogas formationrate. In addition, this data was used to estimate the parameters of theAndrew's equation for the methanogens, which revealed an optimal carbondioxide concentration in the biogas of about 0.5 to about 5% for rapidgrowth. Operation of anaerobic digesters at optimal gas phase carbondioxide concentration will improve the rate of biogas formation. It wasalso found that all autotrophic anaerobic bacteria and archaea appear tohave similar optimal dissolved CO₂ concentrations that are between about0.5% to about 1.0% CO₂(g). High temperature (i.e., thermophilic)anaerobic digestion at low SRT may be attractive, since the rates ofhydrolysis of sewage sludges are much faster than mesophilic operationand may yield an acceptable endproduct (biosolids) with excellentvolatile solids destruction. Alternatively, low temperature anaerobicdigestion may also be possible, since the specific growth rate ofmethanogens is sensitive to temperature. The combination of optimal gasphase carbon dioxide concentration and lower temperature would offer anopportunity for lower capital and operating costs. This discovery goesbeyond municipal wastewater sludge treatment and will also benefit theagriculture industry, which has substantial crop and animal waste thatcould benefit from an anaerobic digester with lower capital costs.

This new process requires a much smaller tank and does not sacrificeperformance as compared to standard anaerobic digestion processes.Resulting equipment costs and capital costs are reduced considerably.Land requirements are similarly reduced.

Also presented is a method and system for sludge treatment in which theCO₂ stripper is repositioned to recycle biogas from the headspace andlower the CO₂ levels before returning it to the headspace or using itdirectly for gas mixing of the bioreactor contents. The repositioning ofthe CO₂ stripper allows for controlling the soluble CO₂ levels to theoptimum CO₂ concentration which increases the specific growth rate ofthe methanogens. This increase results in a reduction in capital costsfor wastewater treatment facilities.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a graph showing the specific growth rate of Anammox bacteriafor low % CO₂ and 24° C.

FIG. 2 is a diagram showing new wastewater treatment plant configurationutilizing Anammox bacteria that grow rapidly with optimal sCO₂.

FIG. 3 is a diagram showing a new wastewater treatment plantconfiguration utilizing Anammox bacteria that grow rapidly with optimalsCO₂ and providing phosphorus removal. In this configuration, the CO₂ isstripped from the primary effluent and combined with the primary sludgeand return activated sludge (RAS) and treated anaerobically forphosphorus release and volatile fatty acid uptake and storage aspolyhydroxybutryate (PHB) or other organic storage polymer by thephosphorus accumulating organisms (PAO).

FIG. 4 is a diagram showing an alternative new wastewater treatmentplant configuration utilizing Anammox bacteria that grow rapidly withoptimal sCO₂ and providing phosphorus removal through use of the primaryclarifier/CO₂ stripping unit process for the phosphorous accumulatingorganism phosphorous release (PAO P-release).

FIG. 5 is a graph showing a comparison of model predictions andexperimental data for anaerobic digester with initial headspaceconcentrations of 0% CO₂ and 100% N₂.

FIG. 6 is a graph showing a comparison of model predictions andexperimental data for anaerobic digester with initial headspaceconcentrations of 20% CO₂ and 80% N₂.

FIG. 7 is a graph showing a comparison of model predictions andexperimental data for anaerobic digester with initial headspaceconcentrations of 50% CO₂ and 50% N₂.

FIG. 8 is a graph showing Andrew's equation for CO₂-reducing methanogensin anaerobic digesters operated at 35° C. As shown in the graph,improved operating conditions are observed when the optimal gas CO₂concentrations are between about 1% to about 10%.

FIG. 9 is a diagram of a standard anaerobic digestion processes for aconventional 2-Stage Anaerobic Digester.

FIG. 10 is a diagram of a standard anaerobic digestion processes for ahigh-rate anaerobic digester.

FIG. 11 is a diagram of the improved anaerobic digestion processes for aconventional 2-Stage Anaerobic Digester.

FIG. 12 is a diagram of the improved anaerobic digestion processes for ahigh-rate anaerobic digester.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments by which the invention may bepracticed. It is to be understood that other embodiments may be utilizedand structural changes may be made without departing from the scope ofthe invention.

All numerical designations, such as pH, temperature, time,concentration, and molecular weight, including ranges, areapproximations which are varied up or down by increments of 1.0 or 0.1,as appropriate. It is to be understood, even if it is not alwaysexplicitly stated that all numerical designations are preceded by theterm “about”. It is also to be understood, even if it is not alwaysexplicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art and can besubstituted for the reagents explicitly stated herein.

The anaerobic autotrophic microorganisms are found in the Bacteria andArchaea branches of the Tree of Life. Several types of anaerobicautotrophic microbes including the Anammox bacteria, sulfate reducingbacteria (SRB), acetogens, dehalogenating bacteria, and methanogens havevalue for environmental remediation, but have limited application due totheir slow specific growth rate or doubling time that is often reportedon the order of days.

Anammox Bacteria

The Anammox bacteria are autotrophic, which means they use dissolved CO₂as their carbon source for growth. Previous research has revealed thatthe dissolved CO₂ concentration can be optimized for another autotrophicbacteria, the nitrifying bacteria, used in the biological treatment ofammonium-rich wastewater. In addition, full-scale wastewater treatmentplants operate at elevated dissolved CO₂ concentrations, which reducethe specific growth rate of the nitrifying bacteria. The impact ofelevated dissolved CO₂ concentrations on the growth of the Anammoxbacteria has not previously been researched.

Rapid growth of Anammox bacteria was discovered by using the FISH methodto identify, enumerate, and evaluate the specific growth rate. Briefly,the FISH method was conducted with a probe designed to detect Anammoxbacteria and it was determined that high levels (>50%) of Anammox werepresent in the bioreactor. The whole cell fluorescence was very highcompared to the cells in the seed material (˜1% abundance), whichindicated high ribosome content in the Anammox bacteria cells of theexperimental bioreactor. This high ribosome content is associated with ahigh specific growth rate.

The specific growth rate or doubling time of the Anammox bacteria wasdetermined to be sensitive to the dissolved CO₂ concentration.Optimizing the dissolved CO₂ concentration increases the specific growthrate (or reduces the doubling time) of the Anammox bacteria. With fasterrates of growth, an alternative for biological ammonium removal may bepossible at full-scale wastewater treatment plants that provide optimaldissolved CO₂ concentrations for the growth of the Anammox bacteria. Thefaster rates of growth may also be used to provide an alternative sludgetreatment system in which a CO₂ stripping process is used where biogasfrom the headspace of an anaerobic digester is flowed through a CO₂stripper and optimized. The optimized CO₂ is then recycled back into thedigester headspace. This results in the use of reactors having a smallerreactor size thus reducing capital and energy costs.

Previous Research

As disclosed in U.S. Pat. Nos. 7,655,143 and 7,641,796 as well as U.S.patent application Ser. No. 12/711,525, each of which is hereinincorporated by reference into this disclosure, the inventor haspreviously developed a method of stimulating nitrification at low SRT byelevating the partial pressure of CO₂ (pCO₂) during aeration (U.S. Pat.No. 7,655,143); an anaerobic digestion process for low-solid waste (U.S.Pat. No. 7,641,796); and a method for the reduction and control of pHand soluble CO₂ for optimal nitrification for domestic, industrial andmunicipal wastewater (U.S. patent application Ser. No. 12/711,525).

As disclosed in U.S. Pat. No. 7,641,796, the inventors previously foundthat thickening the solids content in blended sludge from 1-4% to 10-20%allows for a large reduction in reactor volume. This smaller reactor cancontrol the dissolved CO₂ concentration by controlling the pCO₂concentration in the reactor.

As disclosed in U.S. Pat. No. 7,655,143, elevating pCO₂ increases theconcentration of carbon dioxide and lowers the pH which improvesnitrification thus the specific growth rate of nitrifying bacteria issensitive to pCO₂, pH and dissolved oxygen (DO). The dissolved oxygen isa function of the aeration rate.

As disclosed in U.S. patent application Ser. No. 12/711,525, theinventors previously determined that optimizing the soluble CO₂concentration in the aeration basin of an activated sludge systemsignificantly improves the specific growth rate of the nitrifyingbacteria resulting in a reduction of capital and energy costs formunicipalities.

The method described herein is based on the disclosure in U.S. patentapplication Ser. No. 12/711,525 and U.S. Pat. No. 7,655,143 relating tothe optimal dissolved CO₂ for nitrifying bacteria. The inventors havepreviously used a special chamber to control the dissolved CO₂concentration via control of the pCO₂ concentration in the chamber(described in U.S. Pat. No. 7,641,796). The chamber was anaerobic byusing anaerobic N₂ gas to continuously flush the chamber. An oxygen trapwas used to remove any oxygen from an N₂ cylinder and ensured anaerobicconditions in the chamber. Seed material from a local wastewatertreatment plant was severely diluted into synthetic wastewater with 100mg N/L of ammonium and nitrite. A phosphate buffer was used to controlthe pH to near neutral (about 7 to about 7.5).

In the present invention, the anaerobic autotrophic bacteria and archaeawere found to have growth that is also sensitive to soluble carbondioxide (CO₂) that obeys the Andrew's equation and not the normal Monodequation. In most cases, natural and engineered systems have beenoperating with an elevated soluble CO₂ concentration that severelyinhibits the growth of the anaerobic autotrophic microbes. Operation ofengineered systems (i.e., wastewater treatment plants) at optimalsoluble CO₂ concentration will reduce the capital costs of existingsystems and may lead to new designs for improved performance (i.e.,utilization of Anammox bacteria for treatment of wastewater as analternative to nitrifying bacteria).

Beyond wastewater treatment plants, landfills may benefit from theoperation of lower gas phase CO₂ with the goal of optimizing the solubleCO₂ for higher rates of methanogenesis. Beyond engineered systems, theoptimization of soluble CO₂ may also improve the rates of dehalogenationof organic pollutants in soils and sediments. More recently, there hasbeen interest in ex situ and in situ conversion of fossil fuels (i.e.,coal and oil) to natural gas by the introduction of water and nutrients.The optimization of soluble CO₂ may also increase the rate ofmethanogenesis in these types of systems.

Andrew's Equation

Andrew's equation describes the relationship between specific growthrate of anaerobic autotrophs and dissolved carbon dioxide. Threeparameters are used to define Andrew's equation for anaerobicautotrophs: μ_(max), K_(s,CO2), and K_(i,CO2), where μ_(max) is themaximum specific growth rate, h⁻¹; K_(s,CO2) is the saturation constantfor CO₂, mg/L; and K_(i,CO2) is the inhibition constant for CO₂, mg/L.[CO2] is the concentration of CO₂. The specific growth rate (μ_(obs)) isreduced by the decay coefficient (b or k_(d)). The parameters μ_(max),K_(s), K_(i), and b are estimated to best fit the observed specificgrowth rates.

$\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & \left( {{eq}\mspace{14mu} 1} \right)\end{matrix}$

For anaerobic autotrophs, the dissolved CO₂ or soluble CO₂ (sCO₂) is notoptimal with respect to specific growth rate, which limits biotechnologyapplications utilizing these microbes. Two examples of sCO₂ inhibitionof anaerobic autotrophic microbes are provided.

Example 1 Optimizing CO₂ Concentrations and Using Optimized CO₂Concentrations in Wastewater Treatment Systems

Anammox bacteria are thought to have a fastest doubling time of 12 days,but these bioreactors are operated with 5% CO₂ in the headspace. Thiselevated level of gas phase CO₂ dictates the dissolved CO₂ in thesereactors, which inhibits the specific growth rate of these bacteria.

A lab-scale bioreactor was operated in a special chamber with anaerobicconditions (oxygen-free N₂ gas provided continuously at 100 ml/min andadditions of sodium sulfide, a reducing agent) and about 0.6% CO₂ in thegas phase (Coy CO₂ controller). The lab-scale bioreactor was providednon-limiting ammonium and nitrite (100 mg/L as N) and phosphate bufferto ensure a neutral pH (about 7.6 to about 8). The bioreactor was mixedby use of a magnetic stir bar and stir plate. This bioreactor was usedto enrich activated sludge with an estimated initial 0.5% Anammoxbacteria to >95% Anammox bacteria in a few weeks. This enrichment wasused as the inoculum in a batch test for measuring the optical densityof in side-arm flasks with approximately 19 mL of synthetic media and asmall inoculum of this enrichment (1 mL), which maximized the head spaceavailable (345 mL) for N₂ and about 0.6 to about 1% CO₂. The syntheticmedia was equilibrated to the gas phase CO₂ by placing the side-armflask inside of the anaerobic chamber with constant % CO₂. An aquariumpump inside of the chamber was used to ensure proper flushing of theside-arm flask for 15 minutes before sealing the top with a butyl rubberstopper. Next, the reducing agent (0.05 mL of sodium sulfide) andenrichment (1 mL) was added to the side-arm flask and mixed. Opticaldensity (600 nm) of each side-arm flask was measured by use of aSpectronic bench-top unit. In this manner, an estimate of the specificgrowth rate of the Anammox bacteria was possible for static incubationat room temperature (24° C.), which is lower than the operatingtemperature of conventional Anammox bioreactors (35° C.). This lowerincubation temperature will reduce the observed specific growth ratecompared to the observed maximum specific growth rate (i.e., optimaltemperature and sCO₂ concentration). However, the provision of theoptimal (or near optimal) CO₂ concentration may offset the reduction ofthe specific growth rate due to lower temperature. The CO₂ concentrationand the temperature factors are multiplied together to adjust themaximum specific growth rate. Currently, practitioners improve thespecific growth rate of the microbes by optimizing the temperature inthe reactor (which costs money) for anaerobic autotrophs or increasingthe dissolved oxygen in the basin (which costs money) for the aerobicautotrophs like the nitrifying bacteria. Alternatively, the presentinvention improves the specific growth rate by only optimizing thedissolved CO₂ concentration. Conventional Anammox bioreactors thatoperate at elevated temperature may also benefit from operation atoptimal sCO₂ concentration, which will result in maximizing the specificgrowth rates of the Anammox bacteria when substrates and nutrients arenot limiting.

In this experiment, the maximum specific growth rate of about 0.3267hr⁻¹ or approximate 2 hour doubling time (FIG. 1) was measured at a gasphase CO₂ concentration of about 1%. Rapid growth was also observed atlower % CO₂ concentrations. Static OD measurements were observed withina few hours and were probably due to sCO₂ limitations.

The method used to determine the specific growth rates in FIG. 1 arebased on classical method used by microbiologists: First the celldensity or optical density is measured over time and these values areplotted on a semi-log graph paper. If a line is present on the semi-loggraph paper, then the microbes are growing exponentially and the celldoubling time can be determined graphically or through simplemathematics. The predominant microbe present in the sample was theAnammox bacteria and growth conditions for Anammox were provided (i.e.,anaerobic, CO₂, ammonium and nitrite). The observed specific growth ratewas for the temperature of the reactors: room temperature of about 24°C. These values may be adjusted for temperature, but it is notnecessary. For Anammox, practitioners provide higher temperatures (about35° C.) like they do for the methanogens in anaerobic digesters in orderimprove the specific growth rate. The estimated specific growth rateshown here at lower temp and lower CO₂ concentrations are much higherthan normal practice, thus higher temperatures may not be necessary forAnammox. An exponential curve fit was used for the observed data and thespecific growth rate was provided directly into the equation. The highR² values confirm what is obvious from looking at the plots: themicrobes are in exponential growth phase.

These experimental results suggest that the Anammox bacteria can growmuch faster than reported in the literature. Furthermore, these resultsalso suggest that the Anammox bacteria can grow rapidly at suboptimaltemperatures, which means that wastewater treatment plants may bedesigned and operated in a manner that promotes the growth of thesebacteria.

Optimization of sCO₂ in Use in Wastewater Treatment Systems

Configuration 1

In the simplest design, sCO₂ is optimized by stripping CO₂ fromwastewater after aerobic treatment for BOD removal in a first aerationtank (FIG. 2). The low BOD wastewater with optimal sCO₂ would then besplit with one half aerobically treated for nitrite formation at high pHin the AOB reactor. Caustic addition in the AOB reactor would increasethe pH necessary for rapid ammonium oxidation by the ammonium oxidizingbacteria (AOB) to convert the ammonium to nitrite. The nitrite-richwastewater from the AOB reactor can be combined with the ammonium-richwastewater from the first aeration tank and treated in the Anammoxreactor, which would not be aerated (i.e., anaerobic and optimal sCO₂).In the Anammox reactor, a blend of equal parts ammonium and nitrite isconverted under anoxic conditions to nitrogen gas. In thisconfiguration, BOD and nitrogen removal would be accomplished withoutthe use of an internal recycle, which would reduce capital costs ofnecessary basins compared to the Modified Ludzack-Ettinger process.However, additional capital costs for CO₂ stripping would be necessaryand chemical costs (caustic) for pH adjustment for the AOB reactor.

In this configuration, all of the active biomass passes through anaerobic zone, which may kill most or all of the strict anaerobicbacteria that are necessary for providing anaerobic conditions for theAnammox bacteria. The inventor has observed the presence of low levelsof Anammox bacteria in BardenPho 5-stage systems in Florida that treatraw wastewater. However, Anammox bacteria are not present in MLE systemsthat treat primary effluent or raw wastewater. The design engineers usethe primary solids in the BardenPho 5-stage systems in Florida (andelsewhere) for the generation of volatile fatty acids (VFA) for use bythe phosphorus accumulating organisms. It appears to not be commonknowledge that the lack of primary solids entering into the BardenPho5-stage system may lead to difficulty in achieving strict anaerobicconditions in the initial, anaerobic zone. In other words, thetraditional configuration of the BardenPho 5-stage system (and othersystems designed for P-removal that employ an initial anaerobic zone)has difficulties in achieving true anaerobic conditions without primarysolids due to the lack of strict anaerobic bacteria in these systems.The most likely cause of this absence of strict anaerobic bacteria isthe exposure of all biomass to oxygen in the aerobic zone, which killsstrict anaerobic bacteria. The use of primary solids in the BardenPho5-stage system provides bioaugmentation of the system with strictanaerobic bacteria, which can provide reduced conditions forfermentation (i.e., VFA production), PAO uptake of VFA, and Anammoxreaction. The low levels of Anammox bacteria in these systems are mostlikely due to low levels of nitrite available in the anaerobic zone.

If anaerobic conditions are difficult to maintain in the Anammox reactordue to the destruction of strict anaerobic bacteria, then two operatingstrategies may be employed. First, an anaerobic internal recycle(25-100%×Q_(influent)) of the Anammox reactor effluent to the influentof the Anammox reactor. Second, a small flow rate (1%) of primary solidsor raw wastewater may be periodically or continuously provided to theAnammox reactor to ensure strict anaerobic conditions.

The initial seeding of wastewater treatment systems for Anammox shoulduse activated sludge collected from a BardenPho 5-stage system thattreats primary solids. FISH can be used to screen the seed material toensure that Anammox bacteria are present.

Examples of CO₂ stripping devices include, but are not limited to, airstripping towers and trickling filters. A typical air stripping tower isa column equipped with a blower at the bottom of the column. The airstripping tower is vented to allow air and contaminants to vent to theoutside. The blower blows air upward and the air removes thecontaminants, here CO₂, from the water column. The upward air flowcarries the CO₂ out of the venting system. The air stripping tower canbe filled with packing media as in a packed column. Therefore, the airstripping tower removes contaminants from water by cascading the waterover a packing material designed to uniformly disperse the waterthroughout the tower while providing an upward flow of air which is alsodesigned to uniformly disperse the water throughout the tower as well asprovide a supply of air into which the contaminants may dissipate.

The conventional trickling filter utilizes a film of biomass fixed on amedia to remove and aerobically convert organic matter to carbondioxide, water and additional biomass and to oxidize ammonia tonitrates. The fixed media generally consists of rock, plastic or wood.Wastewater is distributed over the biomass fixed to media through anoverhead rotary distributor having generally two to four nozzled arms orspreaders. This insures a relatively even distribution of wastewaterover the fixed biomass and thereby produces a relatively constantloading throughout the filter area.

These technologies have been developed by the chemical and wastewatertreatment industries. Traditionally, the air stripping tower has beenutilized for high mass transfer rates of volatile organic compounds,while the trickling filter has been used as a fixed-film biologicalprocess for polishing secondary effluent. However, these technologieshave not been employed for removal of soluble CO₂ for the expresspurpose of increasing the specific growth rate of Anammox bacteria.

In some wastewater treatment systems, maintaining the optimal solubleCO₂ in the Anammox reactor may be difficult due to CO₂ generation fromthe anaerobic biodegradation of residual BOD or decay of biomass.Instead of a CO₂ stripper that strips soluble CO₂ from the liquid streamprior to treatment in the Anammox reactor; the Anammox reactor iscovered with a gas impermeable membrane or fixed cover. The CO₂collected in the headspace can be stripped and the gas with very low %CO₂ recycled to the headspace. Instead of mechanically mixing thebiomass and wastewater in the Anammox reactor, the headspace gas or therecycle gas (low % CO₂) can be used for gas mixing. Excess gas in theheadspace can be removed by a pressure relief valve. Since heterotrophicbacteria, AOB, and Anammox bacteria are growing rapidly, the solidsresidence time (SRT) of the system does not need to be maintained athigh values typical of systems designed for nitrogen removal. To ensureproper settling of the activated sludge in the secondary clarifier, theSRT should be maintained at a value of about 5 days, which is comparableto a typical activated sludge system designed for BOD removal. Operationat this lower SRT will also ensure that nitrite oxidizing bacteria areat very low concentrations due to the washout pressure.

Configuration 2

In some cases, nitrite addition may be an attractive alternative to theuse of an AOB reactor (FIG. 3). In this configuration, the CO₂ isstripped from the primary effluent and combined with the primary sludgeand return activated sludge (RAS) and treated anaerobically forphosphorus release and volatile fatty acid uptake and storage aspolyhydroxybutryate (PHB) or other organic storage polymer by thephosphorus accumulating organisms (PAO). Next, the BOD is removedaerobically and the PAO take up the phosphorus by aerobicallymetabolizing the PHB or other organic storage polymer. At this point,excess ammonium remains in the wastewater and nitrite is added prior tothe anaerobic reactor for Anammox. Careful measurement of the ammoniumconcentration in the wastewater entering the Anammox reactor will ensurethat the proper amount of nitrite is provided. If nitrite iscost-prohibitive, then pH adjustment and an AOB reactor upstream of theAnammox reactor may be utilized. Similar to the N-removal system (FIG.2), the SRT of this system should be maintained at a value of about 5days to reduce the level of the nitrifying bacteria. If anaerobicconditions are difficult to maintain in the Anammox reactor, then thetwo strategies described for Configuration 1 may be employed.

Configuration 3

Another version (FIG. 4) uses the primary clarifier/CO₂ stripping unitprocess for the PAO P-release. This primary clarifier doesn't provideoptimal solids separation. Instead, the clarified liquid runs throughthe air stripper to adjust the dissolved CO₂, if needed. However,sufficient hydraulic residence time (HRT) in the primary clarifier,usually between about 30 to about 90 minutes, is required to ensureP-release. In some cases, the addition of an anaerobic basin may beneeded for treatment of the primary sludge prior to aerobic treatment.The rest of the treatment system is similar to FIG. 3. If anaerobicconditions are difficult to maintain in the Anammox reactor, then thetwo strategies described in Configuration 1 may be employed.

Example 2 Optimizing CO₂ Concentrations and Using Optimized CO₂Concentrations in Sludge Treatment Systems

CO₂-reducing methanogens have been identified as the rate-limiting stepin anaerobic digesters. Due to their slow specific growth rate(textbooks use a specific growth rate of about 0.35 d⁻¹), anaerobicdigesters are operated with a high safety factor of 5. The combinationof slow specific growth and the safety factor was used for a recommended15 day solids retention time for anaerobic digesters. However, anaerobicdigesters are operated vessels with a confined head space, where thebiogas is often used for mixing the contents. Typical biogas compositionis about 50-60% methane, about 40-50% CO₂, and low levels of H₂O, N₂,H₂S, and H₂. The extremely high concentration of CO₂ in the headspaceinhibits the specific growth rate of the methanogens and anaerobicautotrophic bacteria, such as the sulfate reducing bacteria (SRB) andacetogens.

Lab-scale anaerobic digesters were used to demonstrate the inhibitioneffect of elevated CO₂ in the gas phase on the specific growth rate ofthe methanogens. Anaerobic digester and sewage sludges were collectedfrom the Glendale wastewater treatment plant at Lakeland, Fla. Sewagesludges were refrigerated prior to experimentation. The solids contentof each sludge was provided. Anaerobic digester sludge (257 mL) wasequilibrated in a 500 mL Pyrex bottle with a total volume of 600 mL. Abutyl rubber stopper with an inlet and outlet port and low pressuregauge (1-30 psi) was secured to each bottle with a modified cap. Oxygenfree gas (CO₂, N₂, or mixture of 20% CO₂:80% N₂) was delivered at 300mL/min for 10 minutes to ensure oxygen removal and initial % CO₂. Forlow % CO₂, two flushes and incubation for at least 30 minutes in theshaker was necessary to equilibrate the soluble CO₂ and gas phase CO₂.In other words, operation at low % CO₂ caused soluble CO₂ to transfer tothe gas phase in order to reach equilibrium according to Henry'sconstant. After equilibration of soluble and gas phase CO₂, each reactorwas fed 43 mL of a mixture of sewage sludges (216 mL of primary sludgeand 50 mL of waste activated sludge). This organic loading rate (˜3 g VSfeed/L bioreactor-day) and F:M (0.25) were selected to avoid organicoverload and subsequent inhibition of methanogenesis for mixedconditions.

The headspace was flushed as described and each anaerobic digester wasincubated at 35° C. and shaken at 150 rpm. Pressure measurements foreach reactor were recorded over time (FIGS. 5-7). Gas leakage wasobserved in the low CO₂ reactors. Parameters were estimated for theAndrew's equation for CO₂-reducing methanogens and combined with anestimated initial biomass level of CO₂-reducing methanogens (9% of VS)and assumed 50% CH₄ and CO₂ of biogas generated. Stoichiometry was usedto link biogas generated to biomass yield (10 mL biogas/mg methanogenbiomass). The high estimated parameters for the Andrew's equation wereμ_(max)=130 d⁻¹, K_(s,CO2)=20 mg/L CO₂, and K_(i,CO2)=0.60 (mg/L CO₂)².The low estimated parameters for the Andrew's equation were μ_(max)=12d⁻¹, K_(s,CO2)=20 mg/L CO₂, and K_(i,CO2)=11.0 (mg/L CO₂)². Theseparameters provide a range for the specific growth rate of themethanogens as a function of headspace % CO₂ and both sets of parametersare in good agreement with the experimental data. Monod kineticsparameters from Metcalf & Eddy were used to model the conventionalbiogas generation (μ=0.35 d⁻¹). All models used a decay coefficient(k_(d)) of 0.02 d⁻¹.

The estimated parameters for the Andrew's equation for the methanogensshow agreement with the specific growth rate used by environmentalengineers (FIG. 7). The Andrew's equation for methanogens suggests thathigher rates of biogas formation are possible by reducing the gas phaseCO₂ concentration below 10% (FIGS. 5 and 6 for 0% and 20% CO₂,respectively). FIG. 8 provides guidance on the effect of gas phase CO₂concentrations, and therefore soluble CO₂ concentrations, on thespecific growth rate of the methanogens. Optimal CO₂ concentrations arecalculated at near 1% in the gas phase (FIG. 8). Most practitioners aremore aware of gas phase concentrations of CO₂ (and CH₄) than the solubleCO₂ concentration. The soluble or dissolved CO₂ concentration isdirectly related to the gas phase CO₂ concentration by Henry's constant.By optimizing the gas phase CO₂ that is in contact with the reactorcontents, the gas phase and soluble phase CO₂ concentration adjust to bein equilibrium with the Henry's constant, temperature and headspacepressure, thus the removal of CO₂ from the gas phase will reduce the gasphase and soluble phase CO₂ concentrations.

It is important to note that all microbes are sensitive to pH and theAndrew's equation can be combined with a Monod term for pH that will bemore accurate in describing the specific growth rate.

$\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & \left( {{eq}\mspace{14mu} 2} \right)\end{matrix}$

In the Monod term for pH, [H⁺] represents the proton concentration andK₁ and K₂ represent the pH factor range limits for growth. K₁ representsthe lower pH limit and K₂ represents the upper pH limit. For example, ifthe pH factor is set for a range of pH between about 6 and about 8 thenK₁ would be 10⁻⁶ and K₂ would be 10⁻⁸. For Anammox bacteria thesuggested pH range is between about 6.7 and about 8.3. Methanogens havebeen observed to grow at a very broad pH range of between a pH of about3 to about 9. The methanogens in the anaerobic digesters have a pH rangeof about 6 to about 8, similar to the Anammox bacteria.

For anaerobic digesters that do not generate high levels of volatilefatty acids, neutral pH (about 6.8- about 8) will be the normal rangeand the dissolved CO₂ will be the key parameter for optimal operationand performance. The pH of the solution is impacted by the carbonatesystem and the volatile fatty acids level. In general, the higher gasphase (and therefore soluble) CO₂ concentrations will lower the pH ofthe solution. By stripping gas phase or soluble phase CO₂ from thesystem, the pH should increase.

Rapid growth of SRB with the optimization of the soluble CO₂ may alsopresent an opportunity to treat the sewage sludges in an anaerobicdigester with a very short HRT with the express purpose of generatingthe bulk of the hydrogen sulfide (H₂S). The retention time depends onthe specific growth rate of the SRB. It is generally thought by those inthe field that the SRB outcompete with the methanogens for H₂ based onthermodynamic considerations. The SRB are also autotrophic and growfaster at optimal CO₂ concentrations. A short HRT is only necessary toremove the sulfate or other sulfur containing compounds that can bereduced to hydrogen sulfide. The HRT depends on the maximum specificgrowth rate of the SRB and the sulfate concentration. The H₂S-rich gascould be flared and the bulk of the remaining undigested sewage sludgeswould be available for anaerobic digestion to methane and CO₂. Thisapproach would separate the bulk of the H₂S from the biogas and reducethe corrosion on biogas handling equipment, since H₂S forms sulfuricacid in the moist biogas.

Traditional anaerobic digestion is a stirred, low-solids processperformed in a continuously-stirred tank reactor (CSTR). Both theconventional and high-rate processes use this design configuration, seenin FIGS. 9 and 10. Currently, there is great interest in generatingbiomethane, which has a methane content of 95-98% and a correspondingcarbon dioxide content of 3-5%. In order to generate biomethane fromconventional and high-rate anaerobic digesters, the headspace biogas ispassed through a CO₂ stripping process. The new anaerobic digestionprocess is novel in that it repositions the biogas purification processequipment (CO₂ stripping) to reduce and optimize the headspace gas phaseCO₂ concentration, seen in FIGS. 11 and 12. In the new anaerobicdigestion process, the CO₂ stripping equipment is positioned so that itreceives high CO₂ biogas from the headspace of the digester. This highCO₂ biogas is flowed through CO₂ stripping equipment which lowers theconcentration of CO₂. This biogas with lower % CO₂ is then recycled backinto the headspace of the anaerobic digester which promotes the growthof the anaerobic bacteria. In FIG. 11, the CO₂ stripping process shownis assumed to operate at 90% CO₂ removal efficiency and a headspace CO₂target concentration of 5%, which generates biomethane. The size andflow rate of the CO₂ stripping process (i.e., capital and operatingcosts) is dictated by the biogas quality, biogas generation rate of theanaerobic digestion process, and CO₂ removal efficiency. Alternatively,this biogas with the lower % CO₂ generated by the CO₂ stripper may alsobe used as the feed gas for a gas mixing system. The new inventionimproves the performance of either configuration. By operating at muchlower gas phase CO₂ concentration, the anaerobic digester can be fed athigher organic loading rates (i.e., food and paper wastes) for a givenSRT or operated at lower SRT for a given organic loading rate. The lowerSRT may require other pre- or post-treatments to meet EPA regulationsfor biosolids. Smaller reactor volume also means that different buildingmaterials than pre-stressed concrete can be used. The new process allowsfor standard sized plastic or metallic tanks. Eliminating excessivereactor size drastically reduces the equipment costs, and as a result,overall capital costs.

It should be noted that the lower SRT that the novel process is designedfor is constrained by regulations for class A pathogen reduction (seeEPA 40 CFR Part 503 for regulations for land application of biosolids).The process retention time necessitates the use of an alternative methodfor pathogen reduction like lime treatment of the biosolids. It is alsoimportant to note that the dimensions for the reactor size listed hereare for domestic/municipal sludge treatment only. Applications involvinganimal, crop, and other organic wastes may require smaller digesters,and will also likely have different disposal regulations.

Uses other than Wastewater and Sludge Digestion

In addition to the digestion of municipal wastewater sludge, thistechnology lends itself to a variety of other waste digestion problemsin industries. Other applications include swine manure (instead ofcovered lagoons), poultry waste, crop waste, paper waste, solid wastepre-landfill treatment, cattle waste, industrial waste, high-solidwaste, and low value fossil fuels, such as coal and oil. In these typesof systems, proper control of the headspace CO₂ concentration (i.e.,circulation of the headspace gas through a molecular sieve for gasseparation or caustic reactor) will reduce and optimize the dissolvedCO₂ concentration in the bioreactor. Covers (fixed or membrane) are usedto maintain anaerobic conditions for the collection of biogas. The CO₂stripper is connected to the biogas enclosure, thus allowing for thetransfer of biogas from the headspace into the CO₂ stripper, where thebiogas with lower CO₂ concentration is returned to the headspace. Theaddition of gas phase CO₂ probes can be used with a controller to ensurethat the target gas phase CO₂ concentration is maintained.

In some cases, intimate mixing of anaerobic microbes with the organicsolids may require a dilution of the mixture with anaerobic processwater with optimal dissolved CO₂ and gas-phase CO₂ concentrations. Thiswould be accomplished by adding a small tank using process water priorto the thickening step. After mixing, the process water can be removedby belt-thickening, gravity separation or centrifugation and the highsolids mixture of organic solid waste and anaerobic microbes would beanaerobically digested. The high solids anaerobic digester would beoperated at the desired temperature (ambient, mesophilic or 35° C., orthermophilic or 55° C.) and optimal gas-phase CO₂ concentration. Sincethermophilic methanogens are autotrophic and phylogenetically relativesof the mesophilic methanogens, the CO₂ sensitivity of the specificgrowth will be similar to the mesophilic methanogens. Lower headspaceCO₂ concentrations will provide better growth conditions for thethermophilic methanogens.

The optimal CO₂ concentration will be driven by Andrew's equation aswell as the capital and operating costs of the system. It may be tooexpensive to operate at 1% gas phase CO₂concentration. However, anyreduction in the current CO₂ concentration will allow the anaerobicdigestion process to operate more efficiently. In this configuration,mixing would not be necessary and the anaerobic digester would beoperated in a plug flow with recycle mode. The optimal gas-phase CO₂concentration would be maintained by continuously removing the CO₂ fromthe biogas in the headspace. The biologically generated biogas comingfrom the anaerobic digestion process will contain between about 30% toabout 50% CO₂. The gas phase CO₂ concentration continually increases dueto the small headspace volume and the generation of biogas with high CO₂levels. Continuously operating the CO₂ stripper reduces the CO₂concentration of the processed biogas to near 0%. This “biomethane” isreturned to the headspace, which dilutes the headspace biogasconcentration of CO₂. When the headspace pressure is too high, then someof the biogas from the headspace is removed from the system. The removalof biogas from the headspace is continuous in most full-scale AD, butthe quality of the biogas is poor due to the high concentration of CO₂.The smaller size and cost of the new system lends itself well to theseapplications, since conventional systems are cost and size prohibitivefor the private user.

For bioreactor landfills that recirculate leachate, coal bed to methanesystems that recycle groundwater and add nutrients prior to injectioninto the subsurface, and other subsurface biogenic methane generationsystems, process water (i.e., leachate or groundwater) can be strippedof excess CO₂ by an anaerobic stripping system (i.e., oxygen freestripping gas) or aerobic stripping system combined with an anaerobicsparging system (i.e., oxygen free N₂ gas) prior to injection into thesesubsurface systems. These approaches would effectively shift thedissolved CO₂ concentration away from the conventional system point andtowards the more optimal soluble CO₂ concentration for methanogenesis.

These approaches may also be beneficial for soil bioremediation sites,where groundwater is pumped to the surface for ex situ treatment andrecycled into the subsurface. Dehalogenating bacteria and methanogenswould grow faster in the subsurface when exposed to optimal soluble CO₂concentrations. For conventional landfills that do not utilize leachaterecirculation, the separation of gas phase CO₂ from the collected biogascan offer an opportunity to recycle the treated biogas with low CO₂content into the landfill gas collection system.

The methods described herein may also be used with anaerobic membranebioreactors (AMB). AMBs are placed after the primary clarifier (thisunit process removes the primary sludge from the wastewater) and beforethe aeration tank or basin used for nitrification, and used to treatdomestic and industrial wastewater. However, these reactors do notoperate at the optimal dissolved CO₂ concentration. These AMB haveenclosed headspaces to ensure anaerobic conditions and collect biogas.The biogas from the AMB could be processed to remove the CO₂ inreal-time to achieve the optimal gas-phase CO₂, using the methodsdescribed above. In turn, the biogas with the optimal gas-phase CO₂ maybe used to aerate the AMB contents in order to increase the rate ofmethanogenesis. Typical use of AMB has limitations in the treatment ofdomestic waste water at low temperatures (<15° C.) since lowtemperatures inhibit methanogenesis. Since both temperature anddissolved CO₂ concentration affect the specific growth rate ofmethanogens as a product (i.e., mu=mu max×temp factor×CO₂ factor), theoptimal dissolved CO₂ concentration offsets the inhibition brought on bylow temperature operation, allowing methanogenesis to occur. Thusoperation at a lower temperature is possible since the CO₂ concentrationoffsets the temperature. This approach would lower the BOD content ofthe wastewater, which would reduce aeration costs compared to aconventional activated sludge system. This would also reduce the amountof sludge needed to be treated by the anaerobic digester for additionalcost savings.

The disclosures of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall there between. Now that theinvention has been described,

What is claimed is:
 1. A method of increasing the specific growth rateof anaerobic, autotrophic microbes in a wastewater treatment systemcomprising: optimizing dissolved CO₂ further comprising: establishing anoptimal control value for the soluble CO₂ concentration for wastewater;measuring the concentration of soluble CO₂ for the wastewater; comparingthe measured concentration of soluble CO₂ in the wastewater against theoptimal control value; and adjusting the measured concentration ofsoluble CO₂ to match the optimal soluble CO₂ control value; wherebyoptimizing the soluble CO₂ increases the specific growth rate of thebacteria.
 2. The method of claim 1, wherein the microbes are Anammoxbacteria.
 3. The method of claim 1, wherein the microbes aremethanogens.
 4. The method of claim 1, wherein the optimal control valueis established by Equation 1 below: $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & (1)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; and b or k_(d) is the decaycoefficient.
 5. The method of claim 1, wherein the optimal control valueis established by Equation 2 below: $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & (2)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; b or k_(d) is the decaycoefficient; [H⁺] is the proton concentration; K₁ is the lower pH limit;and K₂ is the upper pH limit.
 6. The method of claim 1, wherein thesoluble CO₂ concentration of the wastewater in the aeration tank isadjusted by passing the wastewater through CO₂ stripping equipment. 7.The method of claim 1, wherein the soluble CO₂ concentration of thewastewater in the aeration tank is adjusted by passing return activatedsludge through CO₂ stripping equipment.
 8. A method of treatingwastewater for biochemical oxygen demand (BOD) and nitrogen removalcomprising: a. flowing wastewater effluent through a first aerationbasin; b. optimizing CO₂ content of the effluent; c. flowing at least aportion of the optimized CO₂ effluent into a second aeration basin; d.flowing the optimized CO₂ effluent from steps (b) and (c) into ananaerobic reactor; e. flowing the optimized CO₂ effluent from theanaerobic reactor into a secondary clarifier; and f. recycling at leasta portion of wastewater in the secondary clarifier by channeling thewastewater into the first aeration basin.
 9. The method of claim 8,wherein the step of optimizing CO₂ content of the wastewater furthercomprises: establishing an optimal control value for soluble CO₂concentration for the wastewater effluent in the first aeration basinwherein the optimal control value is established by Equation (1)$\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & (1)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; and b or k_(d) is the decaycoefficient; or Equation (2) $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & (2)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; b or k_(d) is the decaycoefficient; [H⁺] is the proton concentration; K₁ is the lower pH limit;and K₂ is the upper pH limit; measuring a concentration of soluble CO₂for the wastewater effluent in the first aeration basin; comparing themeasured concentration of soluble CO₂ in the waste water effluent in thefirst aeration basin against the optimal control value; and adjustingthe soluble CO₂ concentration in the first aeration basin to match theoptimal soluble CO₂ control value.
 10. The method of claim 9, whereinthe soluble CO₂ concentration of the wastewater effluent in the firstaeration basin is adjusted by passing the effluent through a CO₂stripping process.
 11. The method of claim 9, wherein the soluble CO₂concentration of the wastewater effluent in the first aeration basin isadjusted by passing return activated sludge through a CO₂ strippingprocess.
 12. The method of claim 8, wherein the second aeration basin isan ammonium oxidizing bacteria (AOB) reactor.
 13. The method of claim12, wherein the AOB reactor has a pH of at least about
 8. 14. The methodof claim 8, wherein the anaerobic reactor is an Anammox reactor.
 15. Themethod of claim 14, wherein anaerobic conditions are maintained in theAnammox reactor by internally recycling Anammox effluent to Anammoxinfluent.
 16. The method of claim 14, wherein anaerobic conditions aremaintained in the Anammox reactor by flowing about 1% of primary solidsor raw wastewater into the Anammox reactor.
 17. A method of treatingwastewater for BOD, nitrogen and phosphorous removal comprising: a.flowing a volume of wastewater through a primary clarifier; b.optimizing CO₂ content of primary effluent exiting the primaryclarifier; c. flowing the primary effluent having optimized CO₂ into ananaerobic basin for phosphorous release; d. flowing effluent from step(c) to an aerobic basin to remove BOD; e. adding nitrite to the effluentfrom step (d); f. flowing the effluent from step (e) into a secondanaerobic basin; g. flowing the effluent from step (f) into a secondaryclarifier; and h. recycling at least a portion of wastewater in thesecondary clarifier to provide return activated sludge to the firstanaerobic basin.
 18. The method of claim 17, wherein the step ofoptimizing CO₂ content of the primary effluent further comprises:establishing an optimal control value for soluble CO₂ concentration forthe primary effluent exiting the primary clarifier wherein the optimalcontrol value is established by Equation (1) $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & (1)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; and b or k_(d) is the decaycoefficient; or Equation (2) $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & (2)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; b or k_(d) is the decaycoefficient; [H⁺] is the proton concentration; K₁ is the lower pH limit;and K₂ is the upper pH limit; measuring a concentration of soluble CO₂for the primary effluent exiting the primary clarifier; comparing themeasured concentration of soluble CO₂ in the primary effluent exitingthe primary clarifier against the optimal control value; and adjustingthe soluble CO₂ concentration of the primary effluent to match theoptimal soluble CO₂ control value.
 19. The method of claim 18, whereinthe soluble CO₂ concentration of the primary effluent exiting theprimary clarifier is adjusted by passing the wastewater through a CO₂stripping process.
 20. The method of claim 18, wherein the soluble CO₂concentration of the primary effluent exiting the primary clarifier isadjusted by passing return activated sludge through a CO₂ strippingprocess.
 21. The method of claim 17, wherein the second anaerobic basinis an Anammox reactor.
 22. The method of claim 21, wherein anaerobicconditions are maintained in the Anammox reactor by internally recyclingAnammox effluent to Anammox influent.
 23. The method of claim 21,wherein anaerobic conditions are maintained in the Anammox reactor byflowing about 1% of primary solids or raw wastewater into the Anammoxreactor.
 24. A method of treating wastewater for BOD, nitrogen andphosphorous removal comprising: a. flowing a volume of wastewaterthrough a primary clarifier wherein phosphorous is removed by allowingthe wastewater to occupy the primary clarifier for a sufficienthydraulic residence time (HRT); b. optimizing CO₂ content of at least aportion of primary effluent exiting the primary clarifier; c. flowingeffluent from steps (a) and (b) to an aerobic basin to remove BOD; d.adding nitrite to the effluent from step (c); e. flowing the effluentfrom step (d) into an anaerobic basin; f. flowing the effluent from step(e) into a secondary clarifier; and g. recycling at least a portion ofwastewater in the secondary clarifier to provide return activated sludgeto the primary clarifier.
 25. The method of claim 24, wherein the stepof optimizing CO₂ content of the primary effluent further comprises:establishing an optimal control value for soluble CO₂ concentration forthe primary effluent exiting the primary clarifier wherein the optimalcontrol value is established by Equation (1) $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & (1)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; and b or k_(d) is the decaycoefficient; or Equation (2) $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & (2)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; b or k_(d) is the decaycoefficient; [H⁺] is the proton concentration; K₁ is the lower pH limit;and K₂ is the upper pH limit; measuring a concentration of soluble CO₂for the primary effluent exiting the primary clarifier; comparing themeasured concentration of soluble CO₂ in the primary effluent exitingthe primary clarifier against the optimal control value; and adjustingthe soluble CO₂ concentration in the primary effluent to match theoptimal soluble CO₂ control value.
 26. The method of claim 25, whereinthe soluble CO₂ concentration of the primary effluent exiting theprimary clarifier is adjusted by passing the wastewater through a CO₂stripping process.
 27. The method of claim 25, wherein the soluble CO₂concentration of the primary effluent exiting the primary clarifier isadjusted by passing return activated sludge through a CO₂ strippingprocess.
 28. The method of claim 24, wherein the anaerobic basin is anAnammox reactor.
 29. The method of claim 28, wherein anaerobicconditions are maintained in the Anammox reactor by internally recyclingAnammox effluent to Anammox influent.
 30. The method of claim 28,wherein anaerobic conditions are maintained in the Anammox reactor byflowing about 1% of primary solids or raw wastewater into the Anammoxreactor.
 31. A wastewater treatment system comprising: an aerobictreatment zone; an anaerobic treatment zone wherein the anaerobictreatment zone is positioned downstream from the aerobic treatment zone;and a CO₂ stripping zone wherein the CO₂ stripping zone is positionedbetween the aerobic and anaerobic treatment zones.
 32. The system ofclaim 31, further comprising a recycling zone wherein at least a portionof wastewater in the anaerobic treatment zone is recycled to re-enterthe aerobic treatment zone.
 33. A wastewater treatment systemcomprising: a CO₂ stripping zone; at least one anaerobic treatment zonewherein the anaerobic treatment zone is positioned downstream from theCO₂ stripping zone; and an aerobic treatment zone wherein the aerobictreatment zone is positioned between the CO₂ stripping zone and the atleast one anaerobic treatment zone.
 34. The system of claim 33, furthercomprising a recycling zone wherein at least a portion of wastewater inthe anaerobic treatment zone is recycled to re-enter the anaerobictreatment zone.
 35. The system of claim 33, further comprising arecycling zone wherein at least a portion of wastewater in the anaerobictreatment zone is recycled to re-enter the CO₂ stripping zone.
 36. Ananaerobic digestion system comprising: at least one anaerobic digesterhaving a biogas headspace; and CO₂ stripping equipment positionedadjacent the biogas headspace of the at least one anaerobic digester;whereby biogas contained in the biogas headspace is cycled through theCO₂ stripping equipment and re-enters the biogas headspace with lowerCO₂ concentration.
 37. A method of treating sludge comprising: flowingsludge through at least one anaerobic digester having a biogasheadspace; cycling headspace biogas from the biogas headspace throughCO₂ stripping equipment to lower CO₂ levels in the headspace biogas; andflowing the lower CO₂ biogas back into the biogas headspace of the atleast one anaerobic digester; whereby the lower CO₂ headspace biogasencourages the growth of autotrophic anaerobic microbes.
 38. The methodof claim 37, wherein the step of optimizing CO₂ content of the biogasfurther comprises: establishing an optimal control value for gaseous CO₂concentration in the headspace of the at least one anaerobic digesterwherein the optimal control value is established by Equation (1)$\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & (1)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; and b or k_(d) is the decaycoefficient; or Equation (2) $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & (2)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; b or k_(d) is the decaycoefficient; [H⁺] is the proton concentration; K₁ is the lower pH limit;and K₂ is the upper pH limit; measuring a concentration of gaseous CO₂for the biogas in the headspace; comparing the measured concentration ofgaseous CO₂ in the biogas in the headspace against the optimal controlvalue; and adjusting the gaseous CO₂ concentration in the headspace tomatch the optimal gaseous CO₂ control value.
 39. The method of claim 38,wherein the gaseous CO₂ concentration in the headspace is adjusted bystripping CO₂ from the biogas.
 40. The method of claim 38, furthercomprising gas-mixing the contents of the at least one anaerobicdigester using the lower CO₂ biogas.
 41. A method of increasing thespecific growth rate of anaerobic, autotrophic microbes in a wastewatertreatment system comprising: optimizing soluble CO₂ further comprising:establishing an optimal control value for the soluble CO₂ concentrationfor wastewater; measuring the concentration of gaseous CO₂ for thewastewater wherein the gaseous CO₂ concentration is in equilibrium withreactor contents; comparing the measured concentration of gaseous CO₂against the optimal control value; and adjusting the measuredconcentration of gaseous CO₂ to match the optimal soluble CO₂ controlvalue; whereby optimizing the soluble CO₂ increases the specific growthrate of the bacteria.
 42. The method of claim 41, wherein the microbesare Anammox bacteria.
 43. The method of claim 41, wherein the microbesare methanogens.
 44. The method of claim 41, wherein the optimal controlvalue is established by Equation 1 below: $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}}} - b}} & (1)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; and b or k_(d) is the decaycoefficient.
 45. The method of claim 41, wherein the optimal controlvalue is established by Equation 2 below: $\begin{matrix}{{\mu\;{obs}} = {{\mu\;\max \times \frac{\left\lbrack {{CO}\; 2} \right\rbrack}{{\left\lbrack {{CO}\; 2} \right\rbrack + {Ks}},{{{CO}\; 2} + \frac{\left\lbrack {{CO}\; 2} \right\rbrack^{2}}{{Ki},{{CO}\; 2}}}} \times \frac{1}{\left( {1 + \frac{\left\lbrack {H +} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack {H +} \right\rbrack}} \right)}} - b}} & (2)\end{matrix}$ wherein μ_(obs) is the specific growth rate; μ_(max) isthe maximum specific growth rate, h⁻¹; K_(s),CO₂ is the saturationconstant for CO₂, mg/L; K_(i),CO₂ is the inhibition constant for CO₂,mg/L; [CO2] is the concentration of CO₂; b or k_(d) is the decaycoefficient; [H⁺] is the proton concentration; K₁ is the lower pH limit;and K₂ is the upper pH limit.
 46. The method of claim 41, wherein thesoluble CO₂ concentration of the wastewater in the aeration tank isadjusted by passing the wastewater through CO₂ stripping equipment. 47.The method of claim 41, wherein the soluble CO₂ concentration of thewastewater in the aeration tank is adjusted by passing return activatedsludge through CO₂ stripping equipment.